The neonatal period is critical for brain development. Neonatal hypothyroidism, if undetected in the first months of life, will usually lead to mental retardation. If the condition is detected and treatment begun early enough, the child will develop normal intelligence in most cases. For this reason screening of all newborns for hypothyroidism is rapidly becoming a routine practice in many places. A common assay for the detection of hypothyroidism in neonates is radioimmunoassay of thyroxine (T4) concentration using samples of dried blood collected on filter paper.
Radioimmunoassay (RIA) is an analytical method which uses an antiserum that reacts specifically with the hormone to be quantitated, a radioactively labeled form of that hormone which also reacts with the antiserum and thus competes with sample hormone for available antibody binding sites, a separation method which allows one to isolate and count either the antibody bound labeled hormone or the free labeled hormone after a competitive reaction, and calibrators which contain various known amounts of the hormone being quantitated. The procedure involves mixing antibody, radioactive hormone, and either unknown or calibrator in each of a number of tubes which are run in the same assay. The hormone in either calibrator or unknown sample in each assay tube then competes with labeled hormone for the available antibody binding sites during an incubation period. The percentage of labeled hormone binding to the antibody diminishes as the concentration of unlabeled hormone in the samples increases. After separation of bound and free hormone, the labeled hormone is counted in order to determine the amount of radioactivity present. By comparing data obtained from tubes which contained calibrators to data obtained from tubes which contained unknowns, the concentration of hormone in the unknowns is determined.
Radioactivity is not the only way of labeling the hormone. Other possibilities include a flourescent label, a luminescent label, and an enzyme label. The more general term which includes all of these labels is "immunoassay."
The sample collection method utilizing blood samples from a heel prick spotted on filter paper and dried is convenience for screening of neonates. It is most widely used for this purpose rather than the assay of serum or plasma which is used for adults and older children. Serum assays are older and have met the test of wide experience. Serum assay is therefore the method of choice for comparison of results in order to validate the relatively new blood spot assays. It is common to compare results obtained using blood spot samples to results obtained using serum samples. However, investigators ordinarily run such validation experiements with adult blood spots and adult serum samples rather than using neonates. This is due to the difficulty in obtaining speciments from neonates for experimental purposes.
Even though blood spot assays generally correlate well with serum assays when adult subjects are used a review of the literature revealed that mean values for neonatal subjects with serum assays are much higher than mean values for blood spot assays. To demonstrate this inconsistency, the following Table was prepared to show the mean value of nine literature studies using serum samples and four studies using blood spot assays.
TABLE I ______________________________________ Mean T4 Serum Age in Type of Conc., ug/dl Days Sample Reference ______________________________________ 15.0 3-5 Serum 1 15.5 1-3 Serum 2 16.2 1-2 Serum 3 16.4 3-4 Serum 4 16.6 3-4 Serum 5 17.2 3 Serum 6 20.2 2 Serum 7 20.7 3 Serum 8 21.9 2 Serum 9 11.0 3-6 Blood Spot 10, 4 12.4 Not Specified Blood Spot 11 12.6 3 Blood Spot 12, 4 12.81 Not Specified Blood Spot 13 ______________________________________
If the mean values from the nine studies using serum RIA in the above table are averaged, we obtain a value of 17.7 ug/dl for neonates in serum assays. The average neonatal T4 serum concentration calculated from the four studies which assayed dried blood spots is only 12.2 ug/dl. The difference in the blood spot assays and the serum assays is dramatic. Serum assays give on the average of 45.1% higher values than blood spot assays, and there is no overlap between blood spot means and serum assay means for the studies in the Table. This indicates that there is some inherent error in one of the two types of methodologies, more significant than expected variations due to differences in potencies of the calibrators or demographic characteristics of the different populations used for each study.
I have now discovered that in the RIA of T4 in neonates using dried blood collected on filter paper as samples, a dramatic increase in assay values for neonatal samples (but not necessarily for adult samples) occurs when the pH is raised to the region of about 9.2 or above, with an upper limit of possible pH being determined by capability of antiserum binding at high pH or stability of reagents. When the assay of this invention is run at a pH of 8.6, which is approximately the pH of all other procedures of which I am aware, the mean value obtained for neonatal samples is about the same as are obtained with other blood spot assays. As discussed above, serum assays give values about 45% higher than this. When the assay of this invention is run at a pH of 9.2 through 10.0, the mean value of T4 concentration for neonatal samples is approximately constant and is within the range of mean values reported for serum assays.
The RIA of this invention includes a determination of the "blank" or "nonspecific binding" in which the antiserum is omitted from two assway tubes in order to determine to what extent radioactivity is absorbed to the precipitate which contains the antibody-bound radioactivity in the calibrator and unknown tubes. At pH 8.6 the blank is over 10% of the total counts added to the tubes, whereas the blank in the higher pH region drops to about 5% (which probably represents mechanical hold-back rather than nonspecific absorption). This reduction in blank is a desirable effect which accompanies the increase in assay values for neonates at pH of at least 9.2.
The present invention provides a substantial improvement in the art, and its widespread adoption is to be anticipated.